The Mechanism Of Targeting CDCP1 Gene Expression Co-activated By BRD4 And CBP/p300 In Castration-Resistant Prostate Cancer

Background:Prostate cancer(PCa)is the second most common malignancy in males and a leading cause of cancer-related death in the world.Androgen deprivation therapy(ADT)suppresses the production of androgens or blocks androgen receptor(AR)signaling,and has been effective at treating patients with metastatic(advanced)PCa and preventing PCa recurrences.However,most patients develop resistance and eventually progress to androgen-independent castration-resistance prostate cancer(CRPC).At this stage,patient median survival is only-18 months.New generation androgen-axis inhibitors including enzalutamide or abiraterone have shown to improve PCa patient prognosis,but they only extend life expectancy by another-2-3 months.Therefore,CRPC remains a clinically challenging late-stage cancer,which requires further molecular and cellular characterization for new biomarkers and risk stratification to develop novel treatments that can prolong survival.CUB domain-containing protein 1(CDCP1)is a type I transmembrane glycoprotein that contains three extracellular CUB domains and acts as a substrate for the SRC family of kinases.The protein is also known as gp140,CD318,SIMA135,or Trask.It is frequently overexpressed in a variety of human cancers,including prostate,lung,colon,pancreatic,breast,renal,hepatocellular carcinoma,chronic myelogenous leukemia(CML)and acute myeloid leukemia(AML).This overexpression correlates with poor patient prognosis for various malignancies.CDCP1 plays an important role in promoting tumor invasion and metastasis,through its downstream-regulation of the tyrosine phosphorylation-dependent cellular signal pathways,such as PI3K/Akt,PKCδ,SRC and ERK/MAPK.In PCa cells,CDCP1 expression is normally suppressed by androgens and remains at a relatively low level.However,in CRPC,androgen deprivation promotes the upregulation of CDCP1 and subsequent activation of Akt and SRC/MAPK pathways,suggesting a potential association of CDCP1 to the progression of CRPC.Therefore,it is imperative to expand mechanistic understanding of CDCP1 gene transcription activation and pathogenic CDCP1-mediated signaling pathway,which will help to develop CDCP1-targeted inhibitors and new therapeutic CRPC treatments.A major epigenetic regulatory mechanism is the dynamic and reversible lysine acetylation of histones,which is mediated by histone acetyltransferases and usually recognized by bromodomains.This process plays an important role in gene transcription activation and is involved in diverse cellular regulation processes;its dysregulation often leads to irregular expression of oncogenes.The histone acetyltransferase paralogues p300 and CREB-binding protein(CBP),and bromodomain and extra-terminal domain(BET)family proteins(BRD2,BRD3,BRD4 and BRDT)are key transcriptional coactivators that facilitate gene expression.Their functions depend on their recruitment to chromatin through the bromodomains.BRD4 and CBP/p300 frequently overexpressed in a variety of human tumors,and played key roles in activating oncogenic networks for cancer progression.Targeting BRD4 and CBP/p300 represent a therapeutic strategy for new treatment of cancers including CRPC.Therefore,developing potent inhibitors to bromodomains of BET or CBP/p300 proteins,signifies an important step towards suppressing oncogenic pathways in CRPC.Objective:To investigate the epigenetic mechanism of CDCP1 overexpression in CRPC.Targeting CDCP1 transcription by small molecule and providing therapeutic strategy for new treatment of CRPC.Methods:Epigenetics regulation of CDCP1 expression.The expression of CDCP1 and transcription cofactors BRD4 and CBP/p300 in PCa was analyzed using WB assay.The correlation between them was analyzed by GEPIA2 database.Determining the role of BRD4 and CBP/p300 in CDCP1 transcription activation.By performing ChIP assay to investigate whether BRD4 and CBP/p300 simultaneously bind to the CDCP1 promoter or enhancer region and act synergistically to promote CDCP1 transcription.To investigate PCa cells growth and the level of CDCP1-associated signaling molecules upon targeting BRD4 and CBP/p300 respectively or simultaneously.Meanwhile,the synergistic effect of JQ1/IBET762 and SGC-CBP30 on PCa cells was analyzed by synergistic effect experiment.The above results were further confirmed using NEO2734,a dual inhibitor,targeting both bromodomains of BRD4 and CBP/p300.The affinity of NEO2734 was analyzed by thermal shift assay and ITC.Finally,the molecular details of NEO2734 binding to bromodomains of BRD4 and CBP were analyzed using X-ray crystal structures.Results:(1)CDCP1 downregulates distinct cellular pathways in different PCa types,and its overexpression in AR-negative CRPC cells is positively associated with BRD4 and CBP/p300 levels;(2)BRD4 and CBP/p300 are enriched at promoter and enhancer regions of CDCP1 and co-regulate the transcriptional expression of CDCP1.Combined knockdown or inhibition of BRD4 and CBP/p300 inhibits CDCP1 expression more effectively than single knockdown or inhibition;(3)NEO2734,a dual-inhibitor of BRD4 and CBP/p300,shows effective potency for inhibiting both AR-positive and AR-negative CRPC cells;(4)NEO2734 shows potent affinity for both bromodomains of BRD4 and CBP/p300.Crystal structures of NEO2734 with BRD4 and CBP bromodomains display that NEO2734 binds to conserved residues in the respective bromodomain pockets;(5)Dual-targeting of BRD4 and CBP/p300 down-regulates the markers of oncogenic pathways,such as EGFR,c-Myc,SRC/p-SRC,ERK/p-ERK,Akt/p-Akt,indicating a potential method for suppressing CRPC cell cycle,invasion and cellular signaling.Conclusions:In this research,for the first time to reveal that the novel mechanism of BRD4 and CBP/p300 co-activate CDCP1 gene transcription in CRPC.Moreover,discovered dualinhibition of BRD4 and CBP/p300 regulated the level of CDCP1-associated signal pathways and new molecular mechanism of inhibited the proliferation of CRPC cells.Providing the fundamental for optimizing and discovering the novel dual-inhibitor of BRD4 and CBP/p300.